AU694954B2 - Purified myceliophthora laccases and nucleic acids encoding same - Google Patents

Abstract

The present invention relates to isolated nucleic acid constructs containing a sequence encoding a Myceliophthora laccase, and the laccase proteins encoded thereby.

Description

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CORRECTED CORRECTED VESON j VERSION** I page 1, description, replaced by correct page 1 PCT (with an updated version of the pamphlet front page) ~~i~r6~1ci INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 6 (11) International Publication Number: WO 95/33836 C12N 15/53, 1/15, A61K 7/13, 7/06, Al D21C 5/00 (C12N 1/15, C12R 1:66) (43) International Publication Date: 14 December 1995 (14.12.95) (21) International Application Number: PCT/US95/06815 (81) Designated States: AM, AU, BB, BG, BR, BY, CA, CN, CZ, EE, FI, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LT, (22) International Filing Date: 31 May 1995 (31.05.95) LV, MD, MG, MN, MX, NO, NZ, PL, RO, RU, SD, SG, SI, SK, TJ, TM, TT, UA, UG, UZ, VN, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, Priority Data: NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, 08/253,781 3 June 1994 (03.06.94) US GN, ML, MR, NE, SN, TD, TG), ARIPO patent (KE, MW, 08/441,146 15 May 1995 (15.05.95) US SD, SZ, UG).

(71) Applicants: NOVO NORDISK BIOTECH, INC. [US/US]; Published 1445 Drew Avenue, Davis, CA 95616-4880 NOVO With international search report.

NORDISK A/S [DK/DK]; Novo Alld, DK-2880 Bagsvard

(DK).

(72) Inventors: BERKA, Randy, 3609 Modoc Place, Davis, CA 95616 BROWN, Stephen, 3708 Miwok Place, Davis, CA 95616 XU, Feng; 1534 Carmel Valley Drive, Woodland, CA 95776 SCHNEIDER, Palle; Rydtoften 43, DK-2750 Bellerup AASLYNG, Dorrit, Anita; Gartnerkrogen 69, DK-3500 Varlese OX- ENBOLL, Karen, Slotsvej 76, DK-2920 Charlottenlund

(DK).

(74) Agents: ZELSON, Steve, T. et al.; Novo Nordisk of North America, Inc., Suite 6400, 405 Lexington Avenue, New York, NY 10174 (US).

(54) Title: PURIFIED MYCELIOPHTHORA LACCASES AND NUCLEIC ACIDS ENCODING SAME (57) Abstract The present invention relates to isolated nucleic acid constructs containing a sequence encoding a Myceliophthora laccase, and the laccase proteins encoded thereby.

I

CORRECTED

VER:SPON C

PCT

under INID Number (54) 'Title", replace the existing text by "PURIFIED MYCELIOPHTHORA LACCASES AND NUCLEIC ACIDS ENCODING SAME" asrCs~-lq~ INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 6 (11) International Publication Number: WO 95/33836 C12N 15/53, 1/15, A61K 7/13, 7/06, Al D21C 5/00// I(C12N 1/15, C12R 1:66) (43) International Publication Date: 14 December 1995 (14,12.95) (21) International Application Number: PCT/US95/06815 (81) Designated States: AM, AU, BB, BG, BR, BY, CA, CN, CZ, EE, FI, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LT, (22) International Filing Date: 31 May 1995 (31.05.95) LV, MD, MG, MN, MX, NO, NZ, PL, RO, RU, SD, SG, SI, SK, TJ, TM, TI, UA, UG, UZ, VN, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, Priority Data: NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, 08/253,781 3 June 1994 (03.06.94) US GN, ML, MR, NE, SN, TD, TG), ARIPO patent (KE, MW, 08/441,146 15 May 1995 (15.05.95) US SD, SZ, UG).

(71) Applicants: NOVO NORDISK BIOTECH, INC. [US/US]; Published 1445 Drew Avenue, Davis, CA 95616-4880 NOVO With international search report.

NORDISK A/S [DK/DK]; Novo All6, DK-2880 Bagsvaerd

(DK).

(72) Inventors: BERKA, Randy, 3609 Modoc Place, Davis, CA 95616 BROWN, Stephen, 3708 Miwok Place, Davis, CA 95616 XU, Feng; 1534 Carmel Valley Drive, Woodland, CA 95776 SCHNEIDER, Palle; Rydtoften 43, DK-2750 Bellerup AASLYNG, Dorrit, Anita; Gartnerkrogen 69, DK-3500 Varlose OX- ENB0T L, Karen, Slotsvej 76, DK-2920 Charlottenlund

(DK).

(74) Agents: ZELSON, Steve, T. et al.; Novo Nordisk of North America, Inc., Suite 6400, 405 Lexington Avenue, New York, NY 10174 (US).

(54) Title: PURIFIED MYCELIOPHTHORA LACCASES AND NUCLEIC ACIDS ENCODING SAME (57) Abstract The present invention relates to isolated nucleic acid constructs containing a sequence encoding a Myceliophthora laccase, and the laccase proteins encoded thereby.

WO95/33836 PCT/US95/06815 PURIFIED MYCELIOPHTHORA LACCASES AND NUCLEIC ACIDS ENCODING SAME Field of the Invention The present invention relates to isolated nucleic acid fragments encoding a fungal oxidoreductase enzyme and the purified enzymes produced thereby. More particularly, the invention relates to nucleic acid fragments encoding a phenol oxidase, specifically a laccase, of a thermophilic ascomycete, Myceliophthora.

Background of the Invention Laccases (benzenediol:oxygen oxidoreductases) are multi-copper-containing enzymes that catalyze the oxidation of phenolics. Laccase-mediated oxidations result in the production of aryloxy-radical intermediates from suitable phenolic substrate; the ultimate coupling of the intermediates so produced provides a combination of dimeric, oligomeric, and polymeric reaction products. Such reactions are important in nature in biosynthetic pathways which lead to the formation of melanin, alkaloids, toxins, lignins, and humic acids. Laccases are produced by a wide variety of fungi, including ascomycetes such as Aspergillus, Neurospora, and Podospora, the deuteromycete Botrytis, and basidiomycetes such as Collybia, Fomes, Lentinus, Pleurotus, Trametes, and perfect forms of Rhizoctonia. Laccase exhibits a wide range of substrate specificity, and each different fungal laccase usually differs only quantitatively from others in its ability to oxidize phenolic substrates.

Because of the substrate diversity, laccases generally have -1- WO 95/33836 PCT/US95/06815 found many potential industrial applications. Among these are lignin modification, paper strengthening, dye transfer inhibition in detergents, phenol polymerization, juice manufacture, phenol resin production, and waste water treatment.

Although the catalytic capabilities are similar, laccases made by different fungal species do have different temperature and pH optima, and these may also differ depending on the specific substrate. A number of these fungal laccases have been isolated, and the genes for several of these have been cloned. For example, Choi et al.(Mol. Plant-Microbe Interactions 5: 119-128, 1992) describe the molecular characterization and cloning of the gene encoding the laccase of the chestnut blight fungus, Cryphonectria parasitica. Kojima et al. Biol. Chem.

265: 15224-15230, 1990; JP 2-238885) provide a description of two allelic forms of the laccase of the white-rot basidiomycete Coriolus hirsutus. Germann and Lerch (Experientia 41: 801,1985; PNAS USA _83: 8854-8858, 1986) have reported the cloning and partial sequencing of the Neurospora crassa laccase gene. Saloheimo et al. Gen.

Microbiol. 1371 1537-1544, 1985; WO 92/01046) have disclosed a structural analysis of the laccase gene from the fungus Phlebia radiata.

Attempts to express laccase genes in heterologous fungal systems frequently give very low yields(Kojima et al., supra; Saloheimo et al., Bio/Technol. 987-990, 1991). For example, heterologous expression of Phlebia radiata laccase in Trichoderma reesei gave only 20 mg per liter of active enzyme(Saloheimo, 1991, supra). Although laccases have great commercial potential, the ability to express the enzyme in significant quantities is critical to their commercial utility. At the present time there are no laccases which are expressed at high levels in commercially -2- _Jr WO 95/33836 PCT/US95/06815 utilized hosts such as Aspergilus. Thus, the need exists for a laccase which can be produced in commercially useful gram per liter or more) quantities. The present invention fulfills such a need.

Summary of the Invention The present invention relates to a DNA construct containing a nucleic acid sequence encoding a Myceliophthora laccase. The invention also relates to an isolated laccase encoded by the nucleic acid sequence. Preferably, the laccase is substantially pure. By "substantially pure" is meant a laccase which is essentially free of other non-laccase proteins.

In order to facilitate production of the novel laccase, the 4 .nvention also provides vectors and host cells cor. sing the claimed nucleic acid sequence, which vectors and -,ost cells are useful in recombinant production of the laccase. The sequence is operably linked to transcription and translation signals capable of directing expression of the laccase protein in the host cell of choice. A preferred host cell is a fungal cell, most preferably of the genus Aspergillus. Recombinant production of the laccase of the invention is achieved by culturing a host cell transformed or transfected with the construct of the invention, or progeny thereof, under conditions suitable for expression of the laccase protein, and recovering the laccase protein from the culture.

The laccases of the present invention are useful in a number of industrial processes in which oxidation of phenolics is required. These processes include lignin manipulation, juice manufacture phenol polymerization and phenol resin production.

Brief Description of the Figures -3i" Y Y- 1 WO 95/33836 PCT/US95/06815 Figure 1 shows a restriction map of a 7.5 EcoRI fragment in pRaMB1. The region hybridizing to the N. crassa laccase gene probe is shaded.

Figure 2 illustrates the nucleotide(SEQ ID NO: 1) and amino acid (SEQ ID NO: 2) sequence of Myceliophthora thermophila laccase. Lower case letters in the nucleotide sequence indicate the position of introns. Putative TATA and CAAT sequences in the promoter region are in boldface and underlined. Consensus lariat structures(PuCTPuAC)within the introns are underlined.

Figure 3 illustrates the construction of plasmid Detiled Description of the Invention Myceliophthora thermophila is a thermophilic Ascomycete originally described by Apinis (Nova Hedwigia 5: 57-78, 1963) and named Sporotrichum thermophile. Subsequent taxonomic -/isions have placed this organism in the genus Chrysosporium (Von Klopotek, A. Arch. Microbiol. 28: 365- 369, 1974) and later to Myceliophthora (Van Oorschot, Persoonia 9: 401-408, 1977). A number of organisms known by other names also appear to belong to this species. These include Sporotrichum cellulophilum Patent No.

4,106,989); Thielavia thermophila (Fergus and Sinden, Can.

J. Botany 47: 1635-1637, 1968); Chrysosporium fergussi and Corynascus thermophilus (Von Klopotek, supra). This species is known as a source of a number of different industrially useful enzymes, such as cellulases, iglucosidase and xylanase (see, Oberson et al., Enzyme Microb. Technol. 14 303-312, 1992; Merchant et al., Biotechnol. Lett. 10: 513-516, 1988; Breuil et al.

Biotechnol. Lett. 8: 673-676, 1986; Gilbert et al., Bioresource Technol. 39: 147-154, 1992). It has now been determined that Myceliophthora produces a neutral pH WO 95/33836 PCTIUS95/06815 laccase, and the gene encoding this laccase can be used to produce large yields of the enzyme in convenient host systems such as Aspergillus.

To identify the presence of a laccase gene in Myceliophthora, a 5' portion of the Neurospora crassa laccase gene(iccl) is used as a probe, under conditions of mild stringency, in southern hybridization of total genomic DNA of different fungal species. An approximately 12 kb laccase specific sequence is detected in the Myceliophthora DNA. The N. crassa fragment is then used to screen about 20,000 plaques of an M. thermophila genomic DNA library in a X EMBL4 bacteriophage cloning vector. Eight plaques strongly hybridize with the probe; from these eight, DNA is isolated from three. Each of these clones contains a 7.5 EcoRI fragment which also hybridizes to the probe (Figure One of the fragments is subcloned into pBR322 to generate plasmid pRaMBI. Using the Iccl probe, the position of the coding region of the clone is determined. The entire M.

thermophila coding region appears to be contained with a 3.2 kb NheI-BglII segment, which is then cloned into pUC119 and sequenced by the primer walking method.

Once the sequence is determined, the positions of introns and exons within the gene is assigned based on alignment of the deduced amino acid sequence to the corresponding N. crassa laccase gene product. From this comparison, it appears that the gene (lccM) of M.

thermophila is composed of seven exons(246, 79, 12, 70, 973, 69 and 411 nucleotides) interrupted by six introns (85, 84, 102, 72, 147, and 93 nucleotides). The coding region, excluding intervening sequences, is very GC-rich(65.5% G+C) and encodes a preproenzyme of 620 amino acids: a 22 amino acid signal peptide, a 25 amino acid propeptide, and a mature laccase comprising 573 amino acids. The sequence of

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WO 95/33836 PCT/US95/06815 the M. thermophila gene and the predicted amino acid sequence is shown in Figure 2 (SEQ ID NOS: 1 and 2).

The laccase gene is then used to create an expression vector for transformation of Aspergillus host cells. The vector, pRaMB5 contains the A. oryzae TAKA-amylase promoter and terminator regions. The construction of pRaME5 is outlined in Figure 3. Aspergillus cells are cotransformed with the expression vector and a plasmid containrnj the pyrG or amdS selectable marker. Transformants are selected on the appropriate selective medium containing ABTS. Laccaseproducing colonies exhibit a green halo and are readily isolatable. Selected transformants are grown up in shake flasks and culture broths tested for laccase activity by the syringaldazine method. Shake flask cultures are capable of producing 0.2 or more g/liter of laccase, and in fermentors, yields of over 1-2 g/liter are observed.

According to the invention, a Myceliophthora gene encoding a laccase can be obtained by methods described above, or any alternative methods known in the art, using the information provided herein. The gene can be expressed, in active form, using an expression vector. A useful expression vector contains an element that permits stable integration of the vector into the host cell genome or autonomous replication of the vector in a host cell independent of the genome of the host cell, and preferably one or more phenotypic markers which permit easy selection of transformed host cells. The expression vector may also include control sequences encoding a promoter, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes. To permit the secretion of the expressed protein, nucleotides encoding a signal sequence may be inserted prior to the coding sequence of the gene. For expression under the direction of control sequences, a laccase gene to be used -6- WO 95/33836 PCTIUS95/06815 according to the invention is operably linked to the control sequences in the proper reading frame. Promoter sequences that can be incorporated into plasmid vectors, and which can direct the transcription of the laccase gene, include but are not limited to the prokaryotic I-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad.

Sci. U.S.A. 75:3727-3731) and the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25). Further references can also be found in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74- 94; and in Sambrook et al., Molecular Cloning, 1989.

The expression vector carrying the DNA construct of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will typically depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the.replication of which is independent of chromosomal replication, e.g. a plasmid, or an extrachromosomal element, miniqhromosome or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

In the vector, the laccase DNA sequence should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA construct of the invention, especially in a bacterial host, are the promoter of the lac operon of E.coli, the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis -7- WO 95/33836 PCT/US95/06815 a-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciensa-amylase (amyQ), or the promoters of the Bacillus subtilis xylA and xylB genes. In a yeast host, a useful promoter is the eno-l promoter. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding A.

oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral a-amylase, A. niger acid stable a-amylase, A. niger or A. awamori glucoamylase (glaA), Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase. Preferred are the TAKA-amylase and glaA promoters.

The expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably connected to the DNA sequence encoding the laccase of the invention.

Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter. The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.

SThe vector may also comprise a selectable marker, e.g.

a gene the product of which complements a defect in the host cell, such as the dal genes from B.subtilis or B.licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Examples of Aspergillus selection markers include amdS, pyrG, argB, niaD, sC, and hygB, a marker giving rise to hygromycin resistance. Preferred for use in -8-

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WO 95/33836 PCT/US95/06815 an Aspergillus host cell are the amdS and pyrG markers of A.

nidulans or A. oryzae. A frequently used mammalian marker is the dihydrofolate reductase (DHFR) gene. Furthermore, selection may be accomplished by co-transformation, e.g. as described in WO 91/17243.

It is generally preferred that tne expression gives rise to a product which is extracellular. The laccases of the present invention may thus comprise a preregion permitting secretion of the expressed protein into the culture medium. If desirable, this preregion may be native to the laccase of the invention or substituted with a different preregion or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective preregions. For example, the preregion may be derived from a glucoamylase or an amylase gene from an Aspergillus species, an amylase gene from a Bacillus species, a lipase or proteinase gene from Rhizomucor miehei, the gene for the a-factor from Saccharomyces cerevisiae or the calf preprochymosin gene. Particularly preferred, when the host is a fungal cell, is the preregion for A. oryzae TAKA amylase, A. niger neutral amylase, the maltogenic amylase form Bacillus NCIB 11837, B. stearothermophilus a-amylase, or Bacillus licheniformis subtilisin. An effective signal sequence is the A. oryzae TAKA amylase signal, the Rhizomucor miehei aspartic proteinase signal and the Rhizomucor miehei lipase signal.

The procedures used to ligate the DNA construct of the invention, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art for instance, Sambrook et al.. Molecular Cloning, 1989).

-9- WO 95/33836 PCTUS95/06815 The cell of the invention either comprising a DNA construct or an expression vector of the invention as defined above is advantageously used as a host cell in the recombinant production of a enzyme of the invention. The cell may be transformed with the DNA construct of the invention, conveniently by integrating the DNA construct in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.

The host cell may be selected from prokaryotic cells, such as bacterial cells. Examples of suitable bacteria are gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis, 'Bacillus 'entus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyces lividans or Streptomyces murinus, or gram negative bacteria such as E.coli. The transformct.ion of the bacteria may for instance be effected by protoplast transformation or by using competent cells in a manner known per se.

The host cell may also be a eukaryote, such as mammalian cells, insect cells, plant cells or preferably fungal cells, including yeast and filamentous fungi. For example, useful mammalian cells include CHO or COS cells. A yeast host cell may be selected from a species of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces WO 95/33836 PCTMUS95/0C0I5 cerevisiae. Useful filamentous fungi may selected from a species of Aspergillus, e.g. Aspergillus oryzae or Aspergillus niger. Alternatively, a strain of a Fusarium species, e.g. F. oxysporum, can be used as a host cell.

Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023. A suitable method of transforming Fusarium species is described by Malardier et al., 1989.

The present invention thus provides a method of producing a recombinant laccase of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the enzyme and recovering the enzyme from the cells and/or culture medium. The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the laccase of the invention. Suitable media are available from commercial suppliers or may be prepared according to published formulae in catalogues of the American 'Iype Culture Collection).

In a preferred embodiment, the recombinant production of laccase in culture is achieved in the presence of an excess amount of copper. Although trace metals added to the culture medium typically contain a small amount of copper, experiments conducted in connection with the present invention show that addition of a copper supplement to the medium can increase the yield of active enzyme many-fold.

Preferably, the copper is added to the medium in soluble form, preferably in the form of a soluble copper salt, such as copper chloride, copper sulfate, or copper acetate. The final concentration of copper in the medium should be in the -11- b WO 95/33836 PCT[US95/06815 range of from 0.2-2mM, and preferably in the range of from 0.05-0.5mM. This method can be used in enhancing the yield of any recombinantly produced fungal laccase, as well as other copper-containing enzymes, in particular oxidoreductases.

The resulting enzyme may be recovered from the medium by conventional procedures including separating the cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, followed by purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like.

Preferably, the isolated protein is about 90% pure as determined by SDS-PAGE, purity being most important in food, juice or detergent applications.

In a particularly preferred embodiment, the expression of laccase is achieved in a fungal host cell, such as Aspergillus. As described in detail in the following examples, the laccase gene is ligated into a plasmid containing the Aspergillus oryzae TAKA a-amylase promoter, and the Aspergillus nidulans amdS selectable marker.

Alternatively, the amdS may be on a separate plasmid and used in co-transformation. The plasmid (or plasmids) is used to transform an Aspergillus species host cell, such as A. oryzae or A. niger in accordance with methods described in Yelton et al. (PNAS USA 81: 1470-1474,1984).

Those skilled in the art will recognize that the invention is not limited to use of the nucleic acid fragments specifically disclosed herein, for example, in Figure 1. It will also be apparent that the invention encompasses those nucleotide sequences that encode the same amino acid sequences as depicted in Figure 1, but which differ from the specifically depicted nucleotide sequences -12- JvL I.I l WO 95/33836 PCT/US95/06815 by virtue of the degeneracy of the genetic code. Also, reference to Figure 1 in the specification and the claims will be understood to encompass both the genomic sequence depicted therein as well as the corresponding cDNA and RNA sequences, and the phrases "DNA construct" and "nucleic acid sequences" as used herein will be understood to encompass all such variations. "DNA construct" shall generally be understood to mean a DNA molecule, either single- or doublestranded, which may be isolated in partial form from a naturally occurring gene or which has been modified to contain segments of DNA which are combined and juxtaposed in a manner which would not otherwise exist in nature.

The Myceliophthora laccase described herein has a particularly high specific activity on a syringaldazine substrate relative to other known ascomycete or deuteromycete extracellular laccases in which such specific activity has been described. The present sequence provides a.

means by which other such ascomycete and/or deuteromycete laccases can also be isolated. Identification and isolation of laccase genes from sources other than those specifically exemplified herein can be achieved by utilization of the methodology described in the present examples, with publicly available ascomycete and deuteromycete strains. In particular, the specific sequence disclosed herein can be used to design primers and/or probes useful in isolating similar laccase genes by standard PCR or southern hybridization techniques. The present invention thus encompasses those ascomycete and deuteromycete laccases which have a specific activity of at least about 30 SOU/mg, and preferably at least about 40 SOU/mg, "SOU" being defined as mole of substrate oxidized per minute as measured with syringaldazine as a substrate, at optimum pH.

In addition, the invention also encompasses other Myceliophthora laccases, including alternate forms of -13- WO 95/33836 PCT/US95/06815 laccase which may be found in M. thermophila and as well as laccases which may be found in other fungi falling within the definition of Myceliophthora as defined by Van Oorschot, 1977, supra. Identification and isolation of laccase genes from sources other than those specifically exemplified herein can be achieved by utilization of the methodology described in the preseent examples, with publicly available Myceliophthora strains. Alternately, the sequence disclosed herein can be used to design primers and/or probes useful in isolating laccase genes by standard PCR or southern hybridization techniques. Other named Myceliophthora species include Myceliphthora hinnulea (Awao et al., Mycotaxon. 16: 436-440, 1983), Myceliophthora vellerea (Guarro et al, Mycotaxon. 23: 419-427, 1985), and Myceliophthora lutea Costatin. Also encompassed are laccases which are synonyms, anamorphs or perfect states of species or strains of the genus Myceliophthora. Strains of Myceliophthora are readily accessible to the public in a number of culture collections, such as ATCC 48102, 48103, 48104 et al.; CBS 117.65, 131.65, 379.65 et al., DSM 1799 thermophila), ATCC 52474, CBS 539.82, 540.82 et al. hinnulea), DSM 62114, CBS 146..50, 147.50, '57.51 et al lutea), and CBS 478.76, 479.76 and 715.84(M. vellerea). The invention also encompasses any variant nucleotide sequence, and the protein encoded thereby, which protein retains at least about an preferably at least 85%, and most preferably at least 90-95% homology with the amino acid sequence depicted in Figure 1, and which qualitatively retains the laccase activity of the sequence described herein. Useful variants within the categories defined above include, for example, ones in which conservative amino acid substitutions have been made, which substitutions do not significantly affect the activity of the protein. By conservative substitution is meant that amino acids of the same class may be substituted WO 95/33836 PCT/US95/06815 by any other of that class. For example, the nonpolar aliphatic residues Ala, Val, Leu, and Ile may be interchanged, as may be the basic residues Lys and Arg, or the acidic residues Asp and Glu. Similarly, Ser and Thr are conservative substitutions for each other, as are Asn and Gln. It will be apparent to the skilled artisan that such substitutions can be made outside the regions critical to the function of' the molecule and still result in an active enzyme. Retention of the desired activity can readily be determined by conducting a standard ABTS oxidation method, such as is described in the present examples.

The protein can be used in number of different industrial processes. These processes include polymerization of lignin, both Kraft and lignosulfates, in solution, in order to produce a lignin with a higher molecular weight.

A neutral/alkaline laccase is a particular advantage in that Kraft lignin is more soluble at higher pHs. Such methods are described in, for example, Jin et al., Holzforschung 456)L: 467-468, 1991; US Patent No. 4,432,921; EP 0 275 544; PCT/DK93/00217, 1992.

The laccase of the present invention can also be used for in-situ depolymerization of lignin in Kraft pulp, thereby producing a pulp with lower lignin content. This use of laccase is an improvement over the current use of chlorine for depolymerization of lignin, which leads to the production of chlorinated aromatic compounds, which are an environmentally undesirable by-product of paper mills. Such uses are described in, for example, Current opinion in Biotechnology 1: 261-266, 1992; J. Biotechnol. 25: 333-339, 1992; Hiroi et al., Svensk papperstidning 5: 162-166, 1976.

Since the environment in a paper mill is typically alkaline, the present laccase is more useful for this purpose than other known laccases, which function best under acidic conditions.

WO 95/33836 PCT/US95/06815 Oxidation of dyes or dye precursors and other chromophoric compounds leads to decolorization of the compounds. Laccase can be used for this purpose, which can be particularly advantageous in a situation in which a dye transfer between fabrics is undesirable, in the textile industry and in the detergent industry. Methods for dye transfer inhibition and dye oxidation can be found in WO 92/01406; WO 92/18683; EP 0495836; Calvo, Mededelingen van de Faculteit Landbouw-wetenschappen/Rijiksuniversitet G'ent.56: 1565-1567, 1991; Tsujino et al., J. Soc. Chem.42: 273-282, 1991.

The laccase is particularly well-suited for use in hair dyeing. In such an application, the laccase is contacted with a dye precursor, preferably on the hair, whereby a controlled oxidation of the dye precursor is achieved to convert the precursor to a dye, or pigment producing compound, such as a quinoid compound. The dye precursor is preferably an aromatic compound belonging to one of three major chemical families: the diamines, aminophenols(or aminonaphthols) and the phenols. The dye precursors can be used alone or in combination. At least one of the intermediates in the copolymerization must be an ortho- or para-diamine or aminophenol(primary intermediate). Examples of such are found in Section IV, below, and include pphenylene-diamine(pPD), p-toluylene-diamine, chloro-pphenylenediamine, p-aminophenol, o-aminophenol.

3,4-diaminotoluene; additional compounds are also described in US Patent No. 3,251,742, the contents of which are incorporated herein by reference. In one embodiment, the starting materials include not only the enzyme and a primary intermediate, but also a modifier(coupler) (or combination of modifiers), which modifier is typically a meta-diamine, meta-aminophenol, or a polyphenol. Examples of modifier -16- "i WO 95/33836 PCT/US95/06815 compounds include m-phenylene-diamine, 2,4-diaminoanisole, a-naphthol, hydroquinone, pyrocatechol, resorcinol. and 4-chlororesorcinol. The modifier then reacts with the primary intermediate in the presence of the laccase, converting it to a colored compound. In another embodiment, the laccase can be used with the primary intermediate directly, to oxidize it into a colored compound. In all cases, the dyeing process can conducted with one or more primary intermediates, eithe. a °a or in combination with one or more modifiers. Amoun components are in accordance with usual commercial amounts for similar components, and proportions of components may be varied accordingly.

The use of this laccase is an improvement over the more traditional use of H 2 0 2 in that the latter can damage the hair, and its use usually requires a high pH, which is also damaging to the hair. In contrast, the reaction with laccase can be conducted at alkaline, neutral or even acidic pH, and the oxygen needed for oxidation comes from the air, rather than via harsh chemical oxidation. The result provided by the use of the Myceliophthora laccase is comparable to that achieved with use of H 2 0 2 not only in color development, but also in wash stability and light fastness. An additional commercial advantage is that a single container package can be made containing both the laccase and the precursor, in an oxygen free atmosphere, which arrangement is not possible with the use of H 2 0 2 The present laccase can also be used for the polymerization of phenolic compounds present in liquids. An example of such utility is the treatment of juices, such as apple juice, so that the laccase will accelerate a precipitation of the phenolic compounds present in the juice, thereby producing a more stable juice. Such -17- WO 95/33836 PCT/US95/06815 applications have been described in Stutz, Fruit processing 7/93, 248-252, 1993; Maier et al., Dt. Lebensmittelrindschau 86(5): 137-142, 1990; Dietrich et al., Fluss. Obst 57(2): 67-73, 1990,.

Laccases such as the Myceliophthora laccase are also useful in soil detoxification (Nannipieri et al., J.

Environ. Qual. 20: 510-517,1991; Dec and Bollag, Arch.

Environ. Contam. Toxicol. 19.: 543-550, 1990).

The invention is further illustrated by the following non-limiting examples.

EXAMPLES

I. ISOLATION OF MYCELIOPHTHORA THERMOPHILA LACCASE GENE A. MATERIALS AND METHODS 1. DNA Extraction and Hybridization analvsis Total cellular DNA is extracted from fungal cells of Myceliophthora thermophila strain E421 grown 24 hours in ml of YEG medium yeast extract, 2% glucose) using the following protocol: mycelia are collected by filtration through Miracloth (Calbiochem) and washed once with 25 ml of TE buffer. Excess buffer is drained from the mycelia which are subsequently frozen in liquid nitrogen. Frozen mycelia are ground to a fine powder in an electric coffee grinder,and the powder added to 20 ml of TE buffer and 5 ml of 20% SDS in a disposable plastic centrifuge tube.

The mixture is gently inverted several times to ensure mixing, and extracted twice with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1). Sodium acetate (3M solution) is added to give a final concentration of 0.3 M and the nucleic acids are precipitated with 2.5 volumes of ice cold ethanol. The tubes are centrifuged at 15,000 x g for 30 minutes and the pellet is allowed to air-dry for minutes before resuspending in 0.5 ml of TE buffer. DNase- -18- WO 95/33836 PCT/US95/06815 free ribonuclease A is added to a concentration of 100gg/ml and the mixture is incubated at 37'C for 30 minutes.

Proteinase K (200pg/ml) is added and each tube is incubated an additional one hour at 37'C. Finally, each sample is extracted twice with phenol:chloroform:isoamyl alcohol before precipitating the DNA with sodium acetate and ethanol. DNA pellets are dried under vacuum, resuspended in TE buffer, and stored at 4'C.

Total cellular DNA samples from transformants and an untransformed control strain are analyzed by Southern hybridization. Approximately 5g of DNA is digested with EcoRI and fractionated by size on a 1% agarose gel. The gel is photographed under short wavelength UV and soaked for minutes in 0.5 M NaOH, 1.5 M NaCl followed by 15 minutes in 1 M Tris-HCl, pH 8, 1.5 M NaC1. DNA in the gel is transferred onto Zeta-Probem hybridization membrane (BioRad Laboratories) by capillary blotting in 20 X SSPE W.

Davis et al., Advanced Bacterial Genetics, A Manual for Genetic Engineering. Cold Spring Harbor Press. 1980) Membranes are baked for 2 hours at 80'C under vacuum and soaked for 2 hours in the following hybridization buffer at with gentle agitation: 5X SSPE, 35% formamide 0.3% SCS, 200gg/ml denatured and sheared salmon testes DNA.

The laccase-specific probe fragment (approx. 1.5 kb) encoding the 5'-portion of the N. crassa Iccl gene is amplified from N. crassa genomic DNA using standard PCR conditions (Perkin-Elmer Cetus, Emeryville, CA) with the following pair of primers: forward primer, CGAGACTGATAACTGGCTTGG reverse primer, ACGGCGCATTGTCAGGGAAGT The amplified DNA segment is first cloned into a TA-cloning vector (Invitrogen, Inc., San Diego, CA), then purified by agarose gel electrophoresis following digestion with EcoRI. The purified probe fragment is radiolabeled by nick translation with a[ 32 P]dCTP(Amersham) -19- WO 95/33836 PCT/US95/06815 and added to the hybridization buffer at an activity of approximately 1 X 106 cpm per ml of buffer. the mixture is incubated overnight at 45'C in a shaking water bath.

Following incubation, the membranes are washed once in 0.2 X SSPE with 0.1% SDS at 45'C followed by two washes in 0.2 X SSPE(no SDS) at the same temperature. The membranes are allowed to dry on paper towels for 15 minutes, then wrapped in Saran Wrap T and exposed to x-ray film overnight at with intensifying screens(Kodak).

2. DNA Libraries and Identification of Laccase Clones Genomic DNA libraries are constructed in the bacteriophage cloning vector X-EMBL4(J.A.Sorge, in Vectors, A Survey of Molecular Cloning Vectors and Their Uses, Rodriguez et al., eds, pp.43-60, Butterworths, Boston, 1988). Briefly, total cellular DNA is partially digested with Sau3A and size-fractionated on low-melting point agarose gels. DNA fragments migrating between 9kb and 23 kb are excised and eluted from the gel using 8-agarase (New England Biolabs, Beverly MA). The eluted DNA fragments are ligated with BamHI-cleaved and dephosphorylated X-EMBL4 vector arms, and the ligation mixtures are packaged using commercial packaging extracts (Stratagene, LaJolla, CA).

The packaged DNA libraries are plated and amplified on Escherichia coli K802 cells. Approximately 10,000-20,000 plaques from each library are screened by plaquehybridization with the radiolabeled 1ccl DNA fragment using the conditions described above. Plaques which give hybridization signals with the probe are purified twice on E. coli K802 cells, and DNA from the corresponding phage is purified from high titer lysates using a Qiagen Lambda kit(Qiagen, Inc., Chatsworth, CA).

3. Analysis of Laccase Genes WO 95/33836 PCT/US95/06815 Restriction mapping of laccase clones is done using standard methods (Lewin, Genes. 2d ed., Wiley Sons, 1985, New York). DNA sequencing is done with an Applied Biosystems Model 373A automated DNA Sequencer (Applied Biosystems, Inc., Foster City, CA) using the primer walking technique with dye-terminator chemistry Giesecke et al., J. Virol. Methods 38: 47-60, 1992). 0: -onucleotide sequencing primers are synthesized on c, Applied Biosystems model 394 DNA/RNA Synthesizer.

B. RESULTS AND DISCUSSION 1. Identification of Laccase Gene Seauence Total cellular DNA samples are prepared from the 0 species Neurospora crassa, Botrytis cinerea, and Myceliophthora. Aliquots of these DNA preparations are digested with BamHI and fractionated by agarose gel electrophoresis. DNA in the gel is blotted to a Zeta-Probe

T

membrane filter (BioRad Laboratories, Hercules,CA) and probed under conditions of mild stringency with a radiolabeled fragment encoding a portion of the N. crassa Iccl gene, as described above. Laccase-specific sequences are detected in the genomes of M. thermophila and the N.

crassa control, but not in the B. cinerea genomic DNA with this probe.

2. Cloning and Characterization of Mvcelioohthora thermoDhila Laccase (MtL) Gene Approximately 20,000 plaques from a M. thermophila genomic DNA library constructed in a X-EMBL4 cloning vector are screened. The library is composed of approximately 10,000 independent clones with inserts ranging in size from 9kb to 23kb. Assuming an average insert size of 10 kb and a total genome size of 4 x 107 bp for M. thermophila, this figure is about 2.5 times the number of clones required to represent the entire genome. Eight plaques are identified -21t WO 95/33836 PCT/US95/06815 that hybridized strongly to the N. crassa laccase gene probe. DNA is isolated from three of these, cleaved with EcoRI and analyzed by agarose gel electrophoresis and Southern hybridization. All three of these clones contain a 7.5 kb EcoRI fragment which hybridized to the laccasespecific probe. One of these EcoRI fragments is subcloned into pBR322 (Bolivar et al., Gene 2: 95-113, 1977) to generate plasmid pRaMB1. A restriction map of this DNA segment is shown in Fig. 1. The position of the laccase coding region on this clone is determined by hybridization with the Iccl gene fragment described above. Based on mapping data obtained, and an estimated size of the laccase protein of approximately 80 kdal, it is reasoned that the entire M. thermophila laccase coding region is contained with a 3.2 kb NheI-BglII segment which is then subcloned into pUC119(Viera and Messing, Methods Enzymol. 153: 3-11, 1987). The nucleotide sequence of this segment is determined using the primer walking method(Giesecke et al., supra). The nucleic acid sequence is shown in Figure 2 and SEQ ID NO: 1.

The deduced amino acid sequence of MtL is obtained on the basis of amino acid sequence homology with the N. crassa laccase. At the amino acid level, these two laccases share approximately 60% sequence identity. Similarity is highest in regions that correspond to the four histidines and one cysteine which are involved in the formation of the trinuclear copper cluster(Perry et al., J. Gen. Microbiol.

139: 1209-1218, 1993; Coll et al. Appl. Environ. Microbiol.

59: 4129-4135, 1993; Messerschmidt et al. J. Mol. Biol. 206: 513-530, 1989). There are 11 potential sites for N-linked glycosylation in the deduced amino acid sequence of MtL.

the first 22 amino acids of MtL appear to comprise a canonical signal peptide with a predicted cleavage following an Ala residue (vonHeijne,J.Mol. Biol. 173:243-251, 1984).

-22- WO 95/33836 PCTIUS95/06815 Although the amino terminal sequence of the native MtL is unknown, the amino terminus of recombinant MtL produced in A. oryzae is blocked with a pyro-glutamate residue.

Enzymatic removal of this residue followed by amino acid sequencing suggests that mature MtL begins with a Gln residue (position 1 in Figure 2; SEQ ID NO: Thus, MtL is apparently synthesized as a 620 amino acid preproenzyme having a 22 amino acid signal peptide and propeptide of residues. Neurospora crassa laccase(NcL) is processed similarly at its amino terminal end. In addition, NcL is also proteolytically processed at its C-terminus, resulting in the removal of 13 amino acids (Germann et al. J. Biol.

Chem. 263: 885-896, 1988). The processing site is contained within the sequence Asp-Ser-Gly-Leu*Arg 55 (where designates the cleavage site). A similar sequence exists near the C-terminal end of MtL(Asp-Ser-Gly-Leu-Lys56 0 suggesting the Myceliophthora enzyme may also be subject to C-terminal processing (Asp-Ser-Gly-Leu*Lys 56 o) which would remove 12 amino acids.

The positions of six introns (85, 84, 102, 72, 147, and 93 nucleotides) wit 4n the Iccl coding region are determined by comparing the deduced amino acid sequence of MtL to that of NcL and by applying the consensus rules for intron features in filamentous fungi (Gurr et al., in Gene Structure in Eukaryotic Nicrobes, J.R. Kinghorn, ed.) pp 93- 139, IRL Press, Oxford, 1987). The 1860 nucleotides of r coding sequence, excluding introns, are rich in guanosine and cytosine (65.5% The codon usage pattern for this o gene reflects the DNA base composition in a strong Sbias(89.7%) for codons ending in G or C.

II. EXPRESSION OF MYCELIOPHTHORA LACCASE IN ASPERGILLUS A. MATERIALS AND METHODS 1. Bacterial and Fungal Host Strains l- I- Ii, WO 95/33836 PCTUS95/06815 Escherichia coli JM101(Messing et al., Nucl. Acids Res.

9:309-321, 1981) is used as a host for construction and routine propagation of laccase expression vectors in this study. Fungal hosts for laccase expression included the Aspergillus niTer strains Bo-1, AB4.1 and AB1.13(Mattern et al., Mol. Gen. Genet. 234: 332-336), as well as a uridinerequiring(pyrG) mutant of the a-amylase-deficienc Aspergillus oryzae strain HowBl04.

2. Plasmids Plasmid pRaMB2 is a pUCll9 derivative which contains a 3.2 kb BglII-NheI fragment of M. thermophila genomic DNA encoding MtL. The vector pMWR is constructed by inserting the A. oryzae TAKA-amylase promoter and terminator elements from pTAKAl7(Christensen et al., Bio/Technol. 1419-1422, 1988; EP 238 023) into pUC18(Yanisch-Perron et al., Gene 33: 103-119, 1985). In this vector, there is a unique Swal site at the end of the promoter element and a single NsiI site at the beginning of the terminator for directional cloning of coding sequences. The cloning vehicle pUC518 is derived by inserting a small linker containing NsiI, Clal, XhoI, and BglII restriction sites between the adjacent BamHI and XbaI sites of pUC118(Vieira and Messing, supra). Plasmid pToC68(WO 91/17243) contains the A. oryzae TAKA-amylase promoter and A. niger glaA terminator, and 91/17243) carries the A. nidulans amdS gene.

3. Construction of Laccase ExDression Vectors The construction strategy for the laccase expression vector pRaMB5 is outlined in Figure 3. The promoter directing transcription of the laccase gene is obtained from the A. oryzae a-amylase (TAKA-amylase) gene (Christensen et al., supra), as well as the TAKA-amylase terminator region.

The plasmid is constructed first by modifying pMWR3 by inserting a small linker which contains an Apal site between -24i WO 95/33836 PCT/US95/06815 the Swal and NsiI sites, creating a plasmid called pMWR3- SAN. PfuI polymera-e-directed PCR (Stratagene, La Jolla, CA) is used to amplify a short DNA segment encoding the portion of MtL, from the start codon to an internal PstI site (approximately 0.5 kb). The forward primer for this PCR reaction is designed to create an EcoRI site just upstream of the start codon. Next, the amplified fragment is digested with EcoRI and PstI[during this step, the EcoRI site is made blunt by treatment with dNTPs and DNA polymerase I(Klenow fragment)] and purified by agarose gel electrophoresis. The 3' portion of the M. thermophila coding region is excised from pRaMB2 as a 2kb PstI-ApaI fragment(this segment also contains approximately 110 bp from the 3'-untranslated region). These two fragments are combined with Swal- and Apal-cleaved pMWR3-SAN in a threepart ligation reaction to generate the laccase expression vector 4. Transformation of Aspercillus host cells Methods for co-transformation of Aspergillus strains are as described in Christensen et al., supra. For introduction of the laccase expression vectors into A.

oryzae HowB 104 pyrG, equal amounts (approximately 5 gg each) of laccase expression vector and one of the following plasmids are used: pPYRG (Fungal Genetics Stock Center, Kansas City, KS) which contains the A. 7nidulans pyrG gene(Oakley et al, Gene 61385-399, 1987); pSO2 which harbors the clones A.

oryzae pyrG gene; pPRYG24 which contains the A. ficuum(=A.

niger)pyrG gene. Protrophic(Pyr+) transformants are selected on Aspergillus minimal medium (Rowlands and Turner, Mol.

Gen. Genet. 126: 201-216, 1973), and the transformants are transformants are screened for the ability to produce laccase on minimal medium containing 1 mM 2,2'-azinobis(3ethylbenzthiazolinesulfonic acid)[ABTS]. Cells which

I:

i-- WO 95/33836 PCTUS95/06815 secrete active laccase oxidize the ABTS, producing a green halo surrounding the colony. Lastly, A. niger Bo-1 protoplasts are co-transformed using equal amounts (approximately 5jpg each) of laccase expression vector and pToC90 which contains the A. nidulans amdS (acetamidase) gene (Hynes et al., Mol. Cell Biol. 1: 1430-1439, 1983.

AmdS+ transformants are selected on Cove minimal medium (Cove, Biochim. Biophys. Acta 113: 51-56, 1966) with 1% glucose as the carbon source and acetamide as the sole nitrogen source and screened for laccase expression on cove medium with 1 mM ABTS.

Analysis of Laccase-Producin Transformants Transformants which produce laccase activity on agar plates are purified twice through conidiospores and spore suspensions in sterile 0.01% Tween-80 are made from each.

The density of spores in each suspension is estimated spectrophotometrically (A 595 nm). Approximately absorbance units of spores are used to inoculate 25 ml of ASPO4 or MY50 medium in 125 ml plastic flasks. The cultures are incubated at 37'C with vigorous aeration (approximately 200 rpm) for four to five days. Culture broths are harvested by centrifugation and the amount of laccase activity in the supernatant is determined using syringaldazine as a substrate. Briefly, 800 .1 of assay buffer (25 mM sodium acetate, pH 5.5, 40 pM CuSo 4 is mixed with 20 p. of culture supernatant nd 60 .1 of 0.28 mM syringaldazine (Sigma Chemical Co., St. Louis, MO) in ETOH. The absorbance at 530 nm is measured over time in a Genesys 5 UV-vis spectrophotometer (Milton-Roy). One laccase unit(LACU) is defined as the amount of enzyme which oxidizes one pmole of substrate per minute at room temperature. SDS-polyacrylamide gel electrophoresis(PAGE) is done using precast 10-27% gradient gels from Novex(San -26- WO 95/33836 PCT/US95/06815 Diego, CA). Protein bands are developed using Coomassie Brilliant Blue(Sigma).

B.RESULTS AND DISCUSSION 1. Expression of Mvcelioohthora laccase Laccase-producing transformants are detected by incorporation of ABTS into selective media. Using pyrG or amdS as the selectable marker, co-transformation frequencies vary from about 30% to 70%. Heterologous expression of MtL appears to be highest in A. oryzae transformants.

Furthermore, production appears to be better in ASPO4 medium compared to MY50, although the reasons for this are unknown.

SDS-PAGE analysis of culture broth samples shows a prominent laccase band at approximately 80 kdal, which is similar to the size of the native enzyme purified from M. thermophila.

Similar analysis of the culture filtrates from A. niger Botransformants indicate that the laccase band is obscured by very intense glucoamylase and acid-stable amylase protein bands. Results are shown in Table 1.

-27- WO 95133836 WO 9533836PCTIUS95/06815 Table 1. MtL exaressipn amoncr selected A. ory'zae and A.

nicier transfornants

I

HOST STRAIN J TRANSFORMANT TRANSFORMING DNAS f MTLACU/ML I ASP04 !4Y50 A. oryzae H-owBlO4 pyrG untransformed RaMB5.15 RaMB5.30 RaMB5.33 RaMBS .108 RaMB5.11l RaNB5.121 .142 none XpRaMB5+pPYRG pRaMB5+pPYRG pRaMB5+pPYRG pRaMB5+PS02 pRaMB5+PSO2 PRaMB5+PS02 pRaMB5+PSO2 0.00 0.85 0.71 0.60 0.68 0.70 0.49 0.54 0.00 0.29 0.87 0.26 0.19 0.17 0.20 0.04 A. Niger Bo-1 untransformed none 0.00 0.00 RaMB5.1 pRaMB5+pToC90 n.d. 0.20 RaN85.25 pRaMB5+pToC90 n.d. 0.09 RaMB5.49 pRaMB5 pToC9O n.d. 0.06 RaNH5.51 pRaMB5+pToC9O n.d. 0.12 RaMB5.53 PRaMB5+pToC9O n.d. 0.21 RaNB5.62 pRaMB5+pToC9O n.d. 0.16 not determined -28- T

_I-

WO 95/33836 PCTIUS95/06815 2. Expression in the presence or absence of excess coOper A 1 ml aliquot of a spore suspension of Aspergillus oryzae transformant HowBl04-pRaMB5.30(approximately 109 spores/ml) is added aseptically to a 500 ml shake flask containing 100 ml of sterile shake flask medium (maltose, MgS04-7H 2 0, 2g/l; KH 2

PO

4 10g/1; K2SO4, 2g/l; CaC1 2 .2H 2 0 g/l; Citric acid, 2g/l; yeast extract, 10g/1; trace metals[ZnS0 4 .7H 2 0, 14.3 g/l; CuSO 4 .5H 2 0, 2.5 g/l; NiCl 2 .6H 2 0, 0.5 g/l; FeSO 4 .7H 2 0, 13.8 g/l, MnSO 4

-H

2 0, 8.5 g/l; citric acid, 3.0 0.5 ml/l; urea, 2g/l, made with tap water and adjusted to pH 6.0 before autoclaving), and incubated at 37'C on a rotary shaker at 200 rpm for 18 hours. 50 ml of this culture is aseptically transferred to a 3 liter fermentor containing 1.8 liters of the fermentor media (MgSO 4 .7H 2 0, 2g/l; KH 2

PO

4 2g/l; citric acid 4g/l; K 2

SO

4 3g/l;CaCl 2 .2H 2 0, 2g/l; trace metals, 0.5 ml/i; pluronic antifoam, lml/l). The fermentor temperature is maintained at 34'C by the circulation of cooling water through the fermentor jacket. Sterile air is sparged through the fermentor at a rate of 1.8 liter/min The agitiation rate is maintained between 600 and 1300 rpm at approximately the minimum level required to maintain the dissolved oxygen level in the culture above 20%. Sterile feed (Nutriose 725[maltose syrup], 225 g/l; urea, 30 g/l; yeast extract, 15 g/l; pluronic antifoam, 1.5 ml/l, made up with distilled water and autoclaved) is added to the fermentor by us.e of a peristaltic pump. The feed rate profile during the fermentation is as follows: 30 g of feed is added initially before inoculation; 0-24 h, 2 g/l h; 24- 48 h, 4 g/l h, 48h-end, 6 g/l.

Copper is made as a 400X stock in water or a suitable buffer, filter sterilized and added aseptically to the tank -29- WO 95/33836 PCT/US95/06815 to a final level of 0.5 mM. The fermentation described above is also conducted without the addition of copper supplement to tha tank medium. Samples for enzyme activity determination are withdrawn and filtered through Miracloth to remove mycelia. These samples are assayed for laccase activity by the LACU assay described above. Laccase activity is found to increase continuously during the course of the fermentation, with a value of approximately LACU/ml achieved after 180 hours in the fermentation containing excess copper. At a specific activity of 22 LACU/mg, this corresponds to 2g/l of recombinant laccase expressed. On the other hand, the maximum laccase activity achieved in the fermentation without copper supplement is approximately 10 LACU/ml after 170 hours, or about 25% of that found in the presence of additional copper.

SIII. PURIFICATION AND CHARACTERIZATION OF MYCELIOPTHORA

LACCASE

A. MATERIALS AND METHODS 1. Materials Chemicals used as buffers and substrates are commercial products of at least reagent grade. Endo/N-glycosidase F and pyroglutamate amino peptidase are purchased from Boehringer Mannheim. Chromatography is performed on either a Pharmacia FPLC or a conventional low pressure system.

Spectroscopic assays are conducted on either a spectrophotometer(Shimadzu PC160) or a microplate reader(Molecular Devices). Britton Robinson(B&R) buffers are prepared according to the protocol described in Quelle, Biochemisches Taschenbuch, H.M. Raven, II. Teil, S.93 u.

102, 1964.

2. Enzymatic Assay Laccase activity is determined by syringaldazine oxidation at 30'C in a 1-cm quartz cuvette. _j' WO 95/33836 PCT/US95/06815 syringaldazine stock solution (0.28 mM in 50% ethanol) and pg sample are mixed with 0.8 ml preheated buffer solution. The oxidation is monitored at 530nm over minutes. The activity is expressed as lmole substrate oxidized per minute. B&R buffers with various pHs are used.

The activity unit is referred to here as "SOU". A buffer of mM sodium acetate, 40 PM CuS0 4 pH 5.5, is also used to determine the activity, which is referred to as LACU, as defined above. 2,2'-azinobis(3-ethylbenzo thiazoline-6sulfonic acid) (ABTS) oxidation assays are done using 0.4 mM ABTS, B&R buffer, pH 4.1, at room temperature by monitoring

AA

405 An ABTS oxidase activity overlay assay is performed by pouring cooled ABTS-agarose(0.05 g ABTS, 1 g agarose, ml H 2 0, heated to dissolve agarose) over a native IEF gel and incubating at room temperature. Thermostability analysis of the laccase(r-MtL) is performed using samples that have 3 SOU activity pre-incubated in B&R buffer, pH 6, at various temperatures. Samples are assayed after a 400-fold dilution into the same buffer at room temperature.

3. Purification from a fermentor broth 3.7 liters of cheese-cloth filtered broth (pH 7.6, 16 mS) is filtered through Whatman #2 filter paper. The broth is concentrated on a Spiral Concentrator (Amicon) with a S1Y100 membrane (MWCO:100) from 3700 ml to 200 ml. The concentrate is adjusted to 0.75 mS by diluting it in water and reconcentrated on SlY100 to 170 ml. The washed and concentrated broth has a dense greenish color.

The broth is frozen overnight at -20*C, thawed the next day and loaded onto a Q-sepharose XK26 column (120 ml), preequilibrated with 10 mM Tris, pH 7.5, 0.7 mS(Buffer The blue laccase band migrates slowing down the column during loading. One group of blue fractions runs through the column after loading and washing by Buffer A. A second group eluted during the linear gradient with Buffer B -31- WO 95/33836 PCT/US95/06815 (Buffer A plus 2 M NaCl). Some brown material with no laccase activity is eluted out later with 1 M NaOH. SDS- PAGE analysis shows that this preparation results in pure laccase.

4. Analyses of amino acid content, extent of alvcosylation, and N-terminal seauence N-terminal sequencing is performed on an ABI 476A sequencer. Total amino acid analysis, from which the extinction coefficient of r-MtL is determined, is performed on a HP AminoQuant instrument. Deglycosylation is done using endo/N-glucosidase F according to the manufacturer's instructions and carbohydrate content is estimated by mobility difference as determined on SDS-PAGE. N-terminus de-blocking with pyroglutamate amino peptidase is carried out according to manufacturer's instructions. About 804g r- MtL is treated with 4 lg peptidase with or without the presence of 1 M urea or 0.1 M guanidine HCl before being blotted on a PVDF membrane for sequencing. About-20 pmol de-blocked protein is obtained and sequenced.

SDS-PAGE and native IEF analysis are performed on either a Novex cell or a Mini Protean II and a Model 111 Mini IEF cells (Bio-Rad). Gel filtration analyses are done on a Sephacryl S-300(Pharmacia), from which the native MW is estimated by using Blue Dextran (2000 kdal), bovine IgG (158 kdal), bovine serum albumin (66 kdal), ovalbumin (45 kdal) and horse heart myoglobin(17 kdal) to calibrate the column.

B. RESULTS AND DISCUSSION 1. Purification and characterization of r-MtL from a fermentor broth From 3.7 1 of fermentor broth, about 2-3 g of r-MtL are isolated. Initial concentration using a membrane with MWCO of 100 kdal removed significant amounts of brown material and small contaminant proteins. The low affinity of r-MtL toward Q-Sepharose matrix equilibrated with 10 ruM Tris, pH -32- WO 95/33836 PCT/US95/06815 facilitates its separation from other more acidic and more tightly bound impurities. As shown by SDS-PAGE, this preparation resulted in essentially pure laccase for the most active fractions located around the peak. Other less active fractions can be further purified on either Mono-Q with a shallower gradient or a gel filtration column, such as S-300, from which the contaminants are separated due to their smaller MW. An overall 18-fold purification and a recovery of 67% are achieved. As discussed below, the existence of two elution bands of r-MtL on Q-Sepharose chromatogram is probably due to a differential glycosylation.

The purified r-MtL shows a MW of 100-140 kdal on S-300 gel filtration and a MW of 85 kdal on SDS-PAGE. The increase of r-MtL mobility on SDS-PAGE after deglycosylation suggests that carbohydrates account for 14% of its total mass. Native IEF shows a major band at pi -4.2 that is active in ABTS overlay assay.

Directly sequencing the N-terminus of the purified r- MtL from samples either in desalted solution or on PVDF membrane are unsuccessful. However, treatment of r-MtL with pyroglutamate amino peptidase yielded a protein with deblocked N-terminus. This suggests the processing of a propeptide during the maturation of r-MtL, a posttranslational event similar to that of N. crassa laccase but not found in other laccases such as Rhizoctonia solani.

The proposed scheme is outlined below.

MKSFISAATLWIVGILTPSVAAAPPSTEPQRDLLVPITEREEAAVKARQQSCNTPS

I<-putative signal peptide-> putative propeptide <-N-terminus The spectrum of the blue r-MtL has absorption maxima at 276 and 589 nm.

-33- WO 95/33836 PCT/US95/06815 The activity of the laccase is tested by using either syringaldazine and ABTS as substrates. Expressed as per Abs 276 or per mg, the laccase has a value of 20 or 45 units for SOU at pH 6.5, respectively. The LACU assay yields a value of 10 or 22 units per Abs 276 or per mg.

The pH profile of r-MtL activity is quite close to that of the wild type, with an optimal pH of 6.5. The upper temperature limit for retaining full activity after a minute preincubation observed for r-MtL is approximately 60'C. The purified r-MtL shows no activity loss over a week storage frozen in Q-sepharose elution buffer at When comparing the two forms of r-MtL obtained from the fermentor broth isolated on Q-Sepharose, there are no significant differences seen in terms of SDS-PAGE, native PAGE, native IEF, S-300 gel filtration, UV-visible spectrum, specific activity towards syringaldazine and ABTS, and deblocked N-terminus sequencing measurements. Likely, the different elution pattern on Q-Sepharose arises from some sort of differential glycosylation.

IV. USE OF MYCELIOPHTHORA LACCASE IN DYEING HAIR The dyeing effect of Myceliophthora laccase is tested on various dye precursors and further on 0.1% pphenylenediamine compared with a number of modifiers.

Materials: Dye precursors: 0.1 p-phenylene-diamine in 0.1 M K-phosphate buffer, 0.1 o-aminophenol in 0.1 M K-phosphate buffer, Enzymes: Recombinant Myceliophthora thermophila laccase, 16 LACU/ml (in final dye solution).

-34- -r .Y WO 95/33836 PCT/US95/06815 EauiDment: Datacolor Textflash 2000 (CIE-Lab) Assessment of the hair color The quantitative color of the hair tresses is determined on a Datacolor Textflash 2000 by the use of CIE-Lab parameters L* ("0"=black and "100"=white) combined with a* ("-"=green and Results: Dveina effect Tresses of blond European hair (1 gram) are used for testing Myceliophthora thermophila laccase in the context of oxidative hair dyeing. p-phenylene diamine and o-aminophenol are used as the dye precursors.

Hair dveing 4 ml dye precursor solution is mixed with 1 ml laccase on a Whirley mixer, applied to the hair tresses and kept at 30 0

C

for 60 minutes. The hair tresses are then rinsed with running water for about 3 minutes, pressed between two fingers, combed, and air dried.

The results of the dyeing effect test are displayed below in Table 1 and 2.

Table 1 o-aminophenol enzyme L* a" Untreated blond hair 70.3 2.3 Laccase 57.7 15.3 =Dlack, 100=wnice -=green, +=red i WO 95/33836 PCT/US95/06815 Table 2 p-phenylenediamine enzyme L* a Untreated blond hair 70.3 2.3 ml laccase 29.1 4.1 L 0=black, 1UO=white -=green, +=red Result of test: From Table 1 and 2 it can be seen that the Myceliophthora thermophila laccase can be used for oxidative dyeing of hair.

Denosit of Biological Materials The following biological materials have been deposited under the terms of the Budapest Treaty with the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center, 1815 University Street, Peoria, Illinois, 61604 on May 25, 1994, and given the following accession number.

Deposit Accession Number E. coli JM101 containing pRaMB5 NRRL B-21261 -36- *1 WO 95/33836 PCT/US95/06815 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Novo Nordisk Biotech, Inc.

STREET: 1445 Drew Avenue CITY: Davis, California COUNTRY: United States of America POSTAL CODE (ZIP): 95616-4880 TELEPHONE: (916) 757-8100 TELEFAX: (916) 75f-0317

APPLICANT:

NAME: Novo Nordisk A/S STREET: Novo Alle CITY: Bagsvard COUNTRY: Denmark POSTAL CODE (ZIP): DK-2880 TELEPHONE: +45 4444 8888 TELEFAX: +45 4449 3256 (ii) TITLE OF INVENTION: PURIFIED MYCELIOPHTHORA LACCASES AND NUCLEIC ACIDS ENCFODING SAME (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Novo Nordisk of North America, Inc.

STREET: 405 Lexington Avenue, Suite 6400 CITY and STATE: New York, New York COUNTRY: U.S.A.

ZIP: 10174-6401 COMPUTER READABLE FORM: jA) MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (EPO) (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: to be assigned FILING DATE: 31-May-1995

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/253,781 FILING DATE: 03-June-1994 (viii) ATTORNEY/AGENT INFORMATION: NAME: Lowney, Karen A.

REGISTRATION NUMBER: 31,274 REFERENCE/DOCKET NUMBER: 4184.204-WO (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 212 867 0123 TELEFAX: 212 867 0298 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 3187 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear 37

-A

1/'

-II

38 (ii) MOLECULE TYPE: DNA (genoic) (ix) FEATURE: NAME/KEY: CDS LOCATION: join(586..831, 917..994, 1079..1090, 1193..1264, 1337..2308, 2456..2524, 2618..3028) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: rCTAGCTTCT

AGTCGGCTAA

TCATCGAGCG

TCCCTGGTGT

GGACCGGCTC

GGGAAGGGGG

CTTTTTCAAC

CTCCTTCTCG

15 CCTGAGCCAC

TGTCTCTCTT

TTGGTCACCG

GCGATCCTCA

AGTGATCTCC

CGCTAGAGAC

CTTTCACCCC

AGAGAAAGGA

ATCGAGAACA

TCGTCGACTT

CTGAGCCACC

TCTATCGAGT

TCGTTTTCGC CCGCCCCCTC ATCTGGTCTT GTGAGGTCAC ACCACCCAGA AGGGAGGGGG GTCGCGGCAT CAGCCTTTTC CGCGTCCTCC GGAGGATTGA GGGGGGAGGG GCGGAAACAT GGAAGTCGTT GGTGTCGGCC GTCTCAGGTT CTCTCTCTCG TTCAACTCAT CATCTTCAGT CGGCTTCCCG GCCCTTCACC

CCTCCTTCAA

GTCCTCCAGC

CATGCGCGCA

ATCACACCGA

GTC .CGATAT

GTTGGATACG

GTAATGTCTA

TCCACACCAA

CAAGTCGTTC

CCCCCTGAGT

AGATGACAGT

TGCTCCAACA

GCACGTCCAC

TTCGGGATGT

AGCTGCGCCC

TAAAACGAGG

GCCAGTC-TTG

ATTGACATTG

I

I

I i, I I 1 I I

I

I

I

I

1

I

ACAAC ATG AAG TCC Met Lys Ser 1 TTC ATC AGC GCC GCG ACG CTT TTG GTG GGC ATT CTC ACC CCT AGC GTT Phe Ile Ser Ala Ala Thr Leu Leu Val Gly Ile Leu Thr Pro Ser Val 5 10 GCT GCT GCC CCT CCA TCC ACC CCT GAG CAG CGC GAC CTG CTC GTC CCG Ala Ala Ala Pro Pro Ser Thr Pro Giu Gin Arg Asp Leu Leu Val Pro 20 25 30 ATC ACG GAG AGG GAG GAG GCA GCC GTG AAG GCT CGC CAG CAG AGC TGC Ile Thr Giu Arg Giu Giu Ala Ala Val Lys Ala Arg Gin Gin Ser Cys 45 AAC ACC CCC AGC AAC CGG GCG TGC TGG ACT GAC GGA TAC GAC ATC AAC Asn Thr Pro Ser Asn Arg Ala Cys Trp Thr Asp Gly Tyr Asp Ile Asn 55 60 ACC GAC TAC GAA GTG GAC AGC CCG GAC ACG GGT Thr Asp Tyr Giu Val Asp Ser Pro Asp Thr Gly 75 GTT GTT CGG CCG Val Vai Arg Pro GTGAGTGCTC TCGTTAATTA CGCTTCGGCG AGTTGCGCAG ATATATTAAA TACTGCAAAC CTAAGCAGGA GCTGACATGC GACAG TAC ACT CTG ACT CTC ACC GAA GTC GAC Tyr Thr Leu Thr Leu Thr Giu Val Asp AAC TGG ACC GGA CCT GAT GGC GTC GTC AAG GAG AAG GTC ATG CTG GTT Asn Trp Thr Gly Pro Asp Gly Val Val Lys Giu Lys Val Met Leu Val 95 100 105 AAC GTACGGCACC CCTTTTCTTG TCCTAGGATC TGGGTGATGT GCGTCGTTGC Asn 831 891 943 991 1044 j COCTGAGAGA GACTGACCGA GCCTTTGGCT GCAG AAT AGT ATA ATC GTAATTAATT Asn Ser Ile Ile 110 ATACCGCCCT GCCTOCAGOA GCCAGCAG CTCGAGAAGG GTATCTGAAG TTAGTCAGGC CTGCTGACOT GACCGGGGOC AACCCACCAT AG GGA CCA ACA ATC TTT GCG GAO Gly Pro Thr Ile Phe Ala Asp 115 TGG GGC GAO ACG ATC CAG GTA ACG GTC ATO AAC AAC OTO GAG ACC AAC Trp Gly Asp Thr Ile Gin Val Thr Val Ile Asn Asn Leu Giu Thr Asn 120 125 130 135 GGO GTATGTCTGC TGCTTGCTOT CTTGCTOTCC TCGTCCGCGA CTAATAATAA Gly TATCAACTCG TGTGGAAAAC AG ACG TCG ATC CAO TGG CAC GGA CTG CAC CAG Thr Ser Ile His Trp His Gly Leu His Gin 140 145 4 4 4 4 4 4 4 44 4 4 4 4 44 4 4 4 4 4 4 4444 4 4

AAG

Lys

ATC

20 Ile

TAC

Tyr

GGO

Gly 195

GAC

Asp 30

GCC

Al a

GAC

Asp

GGC

Gly

CGC

Arg 275

AAC

Asn

GGC

Gly

CCG

Pro

GGG

Gly 180

GTG

Val

ACC

Thr

GAC

Asp

AAO

Asn

GAG

Glu 260

OTG

Leu

CAC

His CTG CAC Leu His GGA GGG Gly Gly TGG TAC Trp Tyr GCO ATT Ala Ile 200 GGC GTG Gly Val 215 GTG GAA Val Glu TTO AAC Phe Asn AAC GTG Asn Val ACG TCG Thr Ser 280 ACC ATO Thr Ile 295 GAO GGO GCC AAO GGT ATC ACO GAG TGC COG 1.100 1160 1213 1261 1314 1366 1414 1462 1510 1558 1606 1654 1702 1750 1798 1846 1894

I

ATG AOG GTO GAO AGO OTC TTO OTO GGO GTO GGO GAG OGC TAC GAT GTO Met Thr Val Ser Leu Phe CV' i Leu Gly 315 Val Giy Gin Arg Tyr Asp Val 320 GTC ATC GAA GCC AGC OGA ACG 000 GGG AAC TAO TGG TTT AAC GTC ACA Val

TTT

Phe

GC

Ala 355

GGC

Gly 000 Pro

GAC

Asp

GTC

Val

GTC

Val 435

AAC

Ile Giu 325 GGO GGC Gly Gly 340 ATC TTC Ile Phe AAG GC Lys Ala GTC GTG Val. Val AAC AOG Asn Thr 405 TGG AAG Trp Lys 420 GTC GAC Val Asp ATT GTC Ala

GGC

Gly

CAC

His

CCG

Pro

GC

Ala 390

OTO

Leu

GTC

Val

TAC

Tyr

GAG

Ser

CTG

Leu

TAO

Tyr

GTC

Val 375

CGC

Arg

GAO

Asp

AAC

Asn

GTC

Val

GTG

Arg

OTO

Leu

GC

Ala 360

GAC

Asp

GAC

Asp

GTC

Val

GGC

Gly

CTC

Leu 440

AAC

Thr

TGC

Cys 345

GGC

Gly

CAC

His

GTG

Val

ACC

Thr

AGC

Ser 425

AOG

Thr

GGA

Asn Tyr TCC AGG Ser Arg GO GGC Gly Gly 365 CTG GAC Leu Asp 380 AGO GGC Ser Gly ACC ACG Thr Thr AAO ATO Asn Ile ACC AGO Thr Ser 445 CAG GTAI Val

COG

Pro

GAO

Asp

OTO

Leu 385

OGG

Arg

OTG

Leu

AGG

Arg

GGG

Thr

GC

Ala

GAG

Giu 370

AAG

Lys 000 Pro

TTO

Phe 000 Pro

TAO

Phe Pro Pro Gly Tyr 450 ~GAAAAA GGGGAOOGOA 1942 1990 2038 2086 2134 2182 2230 2278 2328 2388 2448 2497 2544 2604 2653 2701 2749 2797 Asn Ile Val. Giu Val Asn Gly Ala Asp Gin 455 460 GGGGTGOTGO TGOAAGTAOA OOTTGOTOGO OOTOOTGTTO TTCOTTAATA ACTACOTOC AAOOOTOCCC OOTAATTAAT TOAOTTTAAA GGOOGATOAA GAOTGAOOGA GOCOOCTOTO TTTGOAG TGG TOG TAO TGG TTG ATO GAG AAO GAT 000 GGO GOA COT TTO Trp Ser Tyr Trp Leu Ile Giu Asn Asp Pro Gly Aia Pro Phe 465 470 ACC OTA COG OAT COG ATG CAC OTG CAC GTAAGTTGGA TAOATATATA Thr Leu Pro His Pro Met His Leu His 475 480 TATATATATA TAOATTGOTT TOOTGGOTOG CTCCOTTAAA TAAAATTAAA TAAOOAAAAA TAAOAAAAAA, AAG GGO CAC GAO TTT TAO G7TG OTG GGO OGO TOG 000 GAO Gly His Asp Phe Tyr Vai Leu Gly Arg Ser Pro Asp 485 490 495 GAG TOG OOG GOA TOO AAO GAG OGG CAC GTG TTO GAT COG GOG OGG GAO Giu Ser Pro Ala Ser Asn Giu Arg His Val Phe Asp Pro Ala Arg Asp 500 505 510 GOG GGO OTG OTG AGO GGG GOC AAO OCT GTG OGG OGG GAO GTG AOG ATG Ala Gly Leu Leu Ser Gly Ala Asn Pro Vai Arg Arg Asp Val Thr Met 515 520 525 O- TG COG GOG TTO GGG TGG GTG GTG OTG GOC TTO CGG GCC GAO AAO COG Leu Pro Ala Phe Gly Trp Val Val. Leu Ala Phe Arg Ala Asp Asn Pro 530 535 540

N',

41 GGC GCC TGG CTG TTC CAC TGC CAC ATC GCC TGG Gly Ala Trp Leu Phe His Cys His Ile Ala Trp 545 550 OTG GGC GTO GTC TAC CTC GAG CGC GCC GAC GAC Leu Gly Val Val Tyr Leu Glu Arg Ala Asp Asp 560 565 570 TCG GAC GOC GAC GCC GAO GAC CTC GAC CGC CTC Ser Asp Ala Asp Ala Asp Asp Leu Asp Arg Leu 580 585 OGC TAO TGG CCT ACC AAC CCC TAO CCC AAG TCC Arg Tyr Trp Pro Thr Asn Pro Tyr Pro Lys Ser 595 600 CAC CGO TGG GTO GAG GAG GGC GAG TGG CTG GTO His Arg Trp Val Glu Glu Gly Glu Trp Leu Val 610 615 AGGAAAAAGG AAACAAAGAG GGGGGGGGGG GCTAGTTCCT TTCTTGTCCT TGTGOTGGCG GTTACCCTGG TAAAGGAGAA GGTGTGTGAT CGGGTAAATA TTATCAAGAG ATOT 20 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 620 amino acids TYPE: amino acid TOPOLOGY: linear CAC GTO TOG GGO GGO His Val Ser Gly Giy 555 OTG OGO GGG GCC GTO Leu Arg Gly Ala Val 575 TGO GOC GAO TGG OGO Cys Ala Asp Trp Arg 590 GAO TOG GGO OTO AAG Asp Ser Gly Leu Lys 605 AAG GCG TGAGCGAAGG Lys Ala 620 ATTTTTGCTT TTTTTTTTTG GGGGGCCCCA AGTTCGAGTG 2845 2893 2941 2989 3038 3098 3158 3192 4 4 444' 4 44 4 4 5 (ii) MOLECULE TYPE: protein

I

Met Pro 4 11 30 Leu Asp Arg Asp Gly Ile (xi) SEQUENCE Lys Ser Phe Ile Ser Val Ala Ala 20 Val Pro Ile Thr Ser Cys Asn Thr 50 Ile Asn Thr Asp Pro Tyr Thr Leu Gly Val. Val Lys 100 Pro Thr Ile Phe 115 Asn Asn Leu Giu 130 DESCRIPTION: SEQ ID Ser Ala Ala Thr Leu Ala Pro Pro Ser Thr Glu Arg Giu Glu Ala Pro Ser Asn Arg Ala Tyr Glu Val Asp Ser 70 Thr Leu Thr Glu Val Glu Lys Val Met Leu 105 Ala Asp Trp Gly Asp 120 Thr Asn Gly Thr Ser 135 NO: 2: Leu V Pro G Ala V Cys T Pro A Asp A Val A Thr I le H 1/ 42 His Gin Lys Gly Thr Asn Leu His Asp Gly Ala Asn GJly Ile Thr Giu 145 150 155 160 4 4 4 4 4444 4 4 4 4 4 4 4 444 4444 4 4 4 44 4 44 4444 4444 444444 4 Cys Gin Gly Pro Ser 225 Phe 15 Gly Arg Leu Asn 305 Asp 25 Val Pro Asp 30 Leu 385 Arg Leu Arg Giy Leu 465 His Pro Gin Asn Tyr 210 Ser Ser Giu Leu Vai 290 Aia Vai Thr Ala Giu 370 Lys Pro Phe Pro Tyr 450 Ile Leu Ile Tyr Giy 195 Asp Ala Asp Gly Arg 275 Asn Met Val Phe Al a 355 Gly Pro Asp Val Val 435 Asn Giu His Pro Pro 165 Gly Thr 180 Val Val Thr Asp Asp Giu Asn Val 245 Giu Tyr 260 Leu Ile His Thr Thr Val Ile Giu 325 Gly Gly 340 Ile Phe Lys Ala Vai Val Asn Thr 405 Trp L~ys 420 Val Asp Ile Val Asn Asp Giy His 485 Lys Ser Gly Leu Leu 230 Leu Ala Asn Met Asp 310 Ala Gly His Pro Ala 390 Leu Val Tyr Glu Pro 470 Asp Gly Tyr Ile 200 Val Giu Asn Val Ser 280 Ile Leu Arg Leu Ala 360 Asp Asp Val Gly Leu 440 Asn Aila Tyr Lys 170 Ser Ile Pro Thr Thr 250 Leu Glu Ala Leu Pro 330 Gly Al a Asn Pro Leu 410 Ala Gin Ala Phe Leu 490 Val His Asn Ile Lys 235 Ala Thr Asn Ala Gly 315 Gly Gly Pro Cys Leu 395 Asp Ile Asn Asp Thr 475 Gly Arg Phe Ser Aia 190 Pro Ala 205 Asp Tyr Ser Gly His Pro Gly Arg 270 Phe Gin 285 Met Val Gly Gin Tyr Trp Arg Asn 350 Gly Pro 365 Asp Leu Gly Phe Thr Gly Ile Asp 430 Ser Phe 445 Trp, Ser Pro His Ser Pro Ser Gly Pro Ala 545 Gly Asp Tyr 15 Arg 4 4 4 4 .44 4 4 4 4 44 4 44 4 44 4 4 44 4: 4,4: 4 4 *1

I.

Claims (41)

1. A substantially pure Myceliophthora laccase having an amino acid sequence which is at least about 80% homologous to the amino acid sequence set forth in SEQ ID NO. 2.

2. A Myceliophthora laccase according to claim 1 which has an amino acid sequence which is at least about 85% homologous to the amino acid sequence set forth in SEQ ID NO. 2.

3. A Myceliophthora laccase according to claim 1 which has an amino acid sequence which is at least about 90% homologous to the amino acid sequence set forth in SEQ ID NO. 2.

4. A Myceliophthora laccase according to claim 1 which has an amino acid sequence which is at least about 95% homologous to the amino acid sequence set forth in SEQ ID NO. 2.

5. A Myceliophthora laccase according to claim 1 which has an amino acid sequence set forth in SEQ ID NO. 2.

6. A laccase of any one of claims 1 to 5 which is a Myceliophthora thermophila laccase.

7. A laccase according to any one of claims 1 to 6 having a specific activity of at least on syringaldazine at optimum pH.

8. A substantially pure Myceliophthora laccase, substantially as hereinbefore described with reference to any one of the examples.

9. A DNA construct comprising a nucleic acid sequence encoding a Myceliophthora laccase according to any one of claims 1 to 8.

10. A DNA construct comprising a nucleic acid sequence encoding a Myceliophthora laccase, substantially as hereinbefore described with reference to any one of the examples.

11. A recombinant vector comprising the DNA construct of claim 9 or claim

12. The vector of claim 11 in which the construct is operably linked to a promoter sequence.

13. The vector of claim 12 in which the promoter is a fungal or yeast promoter.

14. The vector of Claim 13 in which the promoter is the TAKA amylase promoter of Aspergillus oryzae. The vector of Claim 13 in which the promoter is the glucoamylase (gluA) promoter of Aspergillus niger or Aspergillus awamori.

16. The vector of any one of claims 11 to 15 which also comprises a selectable marker.

17. The vector of claim 16 in which the selectable marker is selected from the group consisting of amdS, pyrG, argB, niaD, sC, and hygB.

18. The vector of claim 16 in which the selectable marker is the amdS marker of Aspergillus nidulans or Aspergillus oryzae, or the pyrG marker of Aspergillus nidulans, Aspergillus niger, Aspergillus awamori, or Aspergillus oryzae.

19. The vector of claim 16 which comprises both the TAKA amylase promoter of Aspergillus oryzae and the amdS or pyrG marker of Aspergillus nidulans or Aspergillus oryzae. A recombinant host cell comprising a heterologous DNA construct of claim 9 or claim

21. The cell of claim 20 which is a fungal cell.

22. The cell of claim 20 which is an Aspergillus cell.

23. The cell of any one of claims 20 to 22 in which the construct is integrated into the host 40 cell genome. [n:\libc]00873:SAK ~L~-~ll*T 44 l4 4 I

24. The cell of any one of claims 20 to 23 in which the construct is contained on a vector. The cell of any one of claims 20 to 24 in which the nucleic acid sequence encodes a laccase having the amino acid sequence depicted in SEQ ID NO, 2,

26. A method for obtaining a laccase according to any one of claims 1 to 8, which comprises culturing a recombinant host cell comprising a DNA construct containing a nucleic acid sequence encoding the laccase under conditions conducive to expression of the laccase, and recovering the enzyme from the culture.

27. A method for obtaining a laccase, substantially as hereinbefore described with reference to any one of the examples.

28. A method of enhancing yield of active recombinant Myceliophthora laccase which comprises culturing a recombinant host cell comprising a DNA construct containing a sequence encoding a copper containing enzyme, under conditions conducive to expression of the enzyme, in the presence of at least about 0.02mM copper.

29. A method of enhancing yield of active recombinant Myceliophthora laccase, substantially as hereinbefore described with reference to any one of the examples.

30. A method for polymerising a lignin or lignosulfate substrate in solution which comprises contacting the substrate with a laccase according to any one of claims 1 to 8.

31. A method for polymerising a lignin or lignosulfate substrate in solution, substantially as hereinbefore described with reference to any one of the examples.

32. A method for in situ depolymerisation in Kraft pulp which comprises contacting the pulp with a laccase according to any one of claims 1 to 8.

33. A method for in situ depolymerisation in Kraft pulp, substantially as hereinbefore described with reference to any one of the examples.

34. A method for oxidising dyes or dye precursors which comprises contacting the dye with 25 a laccase according to any one of claims 1 to 8. A method for oxidising dyes or dye precursors, substantially as hereinbefore described with reference to any one of the examples.

36. A method for dyeing hair which comprises contacting a Myceliophthora laccase according to any one of claims 1 to 8, in the presence or absence of at least one modifier, with at least one dye precursor, for a time and under conditions sufficient to permit oxidation of the dye precursor to a dye.

37. The method of claim 36 in which the dye precursor is a diamine, aminophenol or a phenol.

38. The method of claim 37 in which the dye precursor is an ortho- or para-diamine or aminophenol.

39. The method of any one of claims 36 to 38, wherein the modifier, when used, is a meta- diamine, a meta-amino-phenol or a polyphenol. The method of any one of claims 36 to 39 in which more than one modifier is used.

41. The method of any one of claims 36 to 40 in which both the modifier used and the dye precursor is a primary intermediate. [n:\libc]00873:RRB J a, a a a 46

42. A dye composition comprising a Myceliophthora laccase according to any one of claims 1 to 8 col, .oed with at least one dye precursor.

43. A dye composition according to claim 42, further comprising at least one primary intermediate and at least one modifier.

44. A dye composition, substantially as hereinbefore described with reference to any one of the examples. A container containing a dye composition according to any one of claims 42 to 44 in an oxygen free atmosphere.

46. A method of polymerising or oxidising a phenolic or aniline compound which comprises lo contacting the phenolic or aniline compound with a Myceliophthora laccase according to any one of claims 1 to 8.

47. A method of polymerising or oxidising a phenolic or aniline compound, substantially as hereinbefore described with reference to any one of the examples. Dated 12 February, 1997 Novo Nordisk Biotech, Inc. Novo Nordisk AS Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON